1. Field of the Invention
The present invention relates to a method for detecting target substances, as well as to tags, DNA, vectors, probes and detection kits for use with such a method. In particular, the present invention relates to a method for detecting target substances using a polypeptide tag forming an α-helix structure, as well as to tags, DNA, vectors, probes and detection kits for use with such a method.
2. Description of the Related Art
Bioimaging, a technique for visualizing a target protein of interest by selectively labeling it with a fluorescent label, is commonly used for real-time monitoring of expression dynamics and functions of proteins (or polypeptides) within or outside cells. Among widely used techniques for bioimaging of proteins are immunostaining with antibodies labeled with various fluorescent dyes (fluorescent-labeled antibodies) and fusion of target-proteins with fluorescent proteins.
One drawback of immunostaining is that the technique requires as many different fluorescent-labeled antibodies as target proteins and such antibodies are expensive and readily lose their activity. Another disadvantage of immunostaining is that it is difficult to distinguish fluorescent-labeled antibodies bound to target proteins from unbound fluorescent-labeled antibodies, which must be washed off to allow the detection of the target proteins, requiring additional labor.
Concerning the fusion with fluorescent proteins, fluorescent proteins can be incorporated into target proteins as desired using techniques commonly used in the field of genetic engineering. Nonetheless, the wavelength range of excitation/emission that can be used in the detection of fluorescence tends to be limited depending on the type of the fused fluorescent protein.
Another disadvantage of fusion technique is that once a fluorescent protein is expressed, it remains visualized until it is decomposed, so that the timing for visualization cannot be selected as desired. Furthermore, many fluorescent proteins used in the fusion technique are relatively large in size and often affect structure and functions of target proteins.
A new technique for fluorescent labeling of target proteins has recently been proposed. In this technique, a peptide tag is fused with a target protein and the tagged target protein is fluorescent-labeled with tag-specific fluorescent probe molecules (The technique is referred to as “labeling method,” hereinafter). Much effort has been devoted to developing suitable tag/probe pairs for use in the labeling method (See, for example, C. Irwin, et al., Curr. Opin. Biotechnol., 2005, 16, 35-40; and A. Ojida, et al., J. Am. Chem. Soc., 2006, 128, 10452-10459). The labeling method enables the use of a variety of fluorescent molecules obtained by organic synthesis. In addition, the method, in which tag-fused proteins are expressed prior to labeling, allows detection of target proteins at any time point and at various excitation/emission wavelengths. However, it is considered difficult to distinguish fluorescent probe molecules bound to the tag-fused proteins from the unbound fluorescent probe molecules, which must be washed off to allow the detection of the target proteins, requiring additional labor.
Leucine zipper is one of the known higher structures of peptide chains found in proteins. It is a higher structure formed by two or more α-helices of peptides that are highly complementary to and, thus, have high affinity for one another. The α-helices are held together through hydrophobic interaction and electrostatic interaction. An α-helix structure is a right-handed helix of amino acids that has 3.6 amino acids in each turn with a pitch of 0.54 nm. Each carbonyl group in the polypeptide chain forms a hydrogen bond with the amide of the amino acid four residues ahead in the polypeptide chain, forming a substantially linear backbone that makes the structure highly stable.
Recent studies report that hydrophobic pockets can be formed within a peptide bundle having a leucine zipper structure consisting of three α-helices by replacing hydrophobic amino acids in the peptide bundle with amino acids having small side chains. Fluorescent dyes and other small organic molecules can be incorporated into such pockets (See, for example, I. Obataya, et al., Biopolymers, 2001, 59, 65-71).